Research

The Leukotriene Synthetic Complex

The leukotrienes (LTs) are bioactive signaling molecules generated in myeloid cells from arachidonic acid (AA) that initiate and amplify innate and adaptive immunity. There are two parent LTs, LTB₄ and LTC₄. LTB₄ initiates and amplifies the innate immune response by functioning as a highly potent chemoattractant for PMNs, monocytes, mast cells, and certain T-cell subsets. LTC₄ and its products LTD₄ and LTE₄ have a variety of effects on airway and vascular smooth muscle. To prevent the unwanted initiation of inflammation and ensure that an inflammatory response is appropriately matched to a given challenge, cells have evolved a complex series of molecular controls to regulate LT synthesis. In resting cells, 5-lipoxygenase (5-LO), the initial enzyme of the LT pathway, resides in the cytosol and nucleoplasm, and cytosolic phospholipase A₂ (cPLA₂) is localized in the cytosol. The substrate AA is esterified to phospholipids, and the generation of free AA by cPLA₂ is required for the oxygenation of AA by 5-LO. To initiate LT synthesis, increased intracellular calcium triggers the translocation of cPLA₂ to the endoplasmic reticulum (ER), outer nuclear membrane, and the Golgi. In parallel, 5-LO translocates to the inner and outer nuclear membranes. The N-terminal C2 domain of cPLA₂ and C2-like domain of 5-LO mediate their association with specific membrane phospholipids. On nuclear membranes, 5-LO must interact with its integral membrane scaffold protein, the 5-lipoxygenase-activating protein (FLAP). FLAP then functions to bring AA into apposition with 5-LO allowing the formation of the initial LT, LTA₄. 5-LO and FLAP form the core of a multiprotein LT synthetic complex. The cores, along with other proteins, are further combined to form supramolecular nanoclusters.

As for many signaling complexes, the transient assembly of the LT synthetic complex on the nuclear membrane is governed by weak interactions between its members. These properties allow the LT synthetic complex to integrate multiple signals to achieve a graded output of LTs and to maintain biological flexibility; however, they also complicate analysis of composition and function. Different combinations of input signals can lead to different complex compositions, post-translational modifications of its members, and supramolecular organization. We are combining molecular imaging, cell biology, and biochemistry to define properties of this structure, identities of novel interacting proteins such as AP-10, and the supramolecular organization of LT synthesis.

Distinct extracellular signals synergize to alter the assembly and output of the LT synthetic complex. Fluorescence lifetime imaging microscopy (FLIM) permits quantitative visualizatio of protein interactions. Using isolated neutrophils, we looked at the interaction of 5-LO and FLAP in response to a weak stimulus (C5a) alone and in combination with a priming factor (GM-CSF), which boosts C5a-dependent LT synthesis. Shorter τ1 values correlate increased LT synthesis with a tighter interaction between 5-LO and FLAP. For more about signal integration with the LT synthetic complex, check out our recent paper.